The applicants have cloned a cDNA sequence coding for a novel EGF repeat-containing protein, which they have designated preadipocyte factor-1 (pref-1). Pref-1 is highly expressed in 3T3-L1 preadipocytes, but its transcription decreases during adipocyte differentiation and pref-1 is not detectable in mature adipocytes. In fact, when constitutively expressed by transfection, pref-1 blocks adipose conversion of 3T3-L1 cells. The goal of this research is to elucidate the molecular mechanisms underlying the expression of pref-1, an inhibitor of adipogenesis, in preadipocytes and its down-regulation during adipose conversion. They will first determine the cis-acting sequences responsible for the high level of pref-1 gene expression in 3T3-L1 preadipocytes by transfecting various pref-1 promoter-luciferase fusion constructs into preadipocytes. Next, they will define the DNA sequences responsible for negative regulation of pref-1 gene during adipose conversion by transfecting these plasmids into preadipocytes and adipocytes and comparing the promoter activities. Moreover, they will identify the trans-acting factors which mediate the expression of pref-1 in preadipocytes and its repression during adipogenesis by in vitro footprinting and gel shift assays. The trans-acting factors will then be purified and/or cloned to further examine pref-1 gene transcription at the molecular level. These studies will enable them to direct the expression of genes to preadipocytes and to identify a regulatory molecule that acts at a stage prior to pref-1 during adipocyte differentiation. In addition, they will examine the factors that affect expression of the pref-1 gene, which in turn regulates adipose conversion process. This will give them the means to alter expression of pref-1 gene in the future.